The present invention pertains in general to compositions and methods for functionalizing nucleic acids and in particular to compositions and methods for the 5' labelling of polynucleotides attached to a support.
Attachment of a detectable label to a polynucleotide permits detection and quantitation of a nucleic acid. The attachment of detectable labels to nucleic acids is particularly important in hybridization assays wherein a labelled polynucleotide probe is used to search a sample for a target nucleic acid which has a complementary nucleotide sequence and which has been immobilized by hybridization to a support-bound polynucleotide probe. Labelling with a reactive group permits attachment of reporter groups to or immobilization of the polynucleotide.
One approach to labelling a polynucleotide probe for use in hybridization assays involves binding a radioisotope (e.g., .sup.32 P, .sup.3 H, or .sup.125 I) to the probe. However, difficulties inherent in the two methods of detecting radioactive labels limit the usefulness of the technique. Autoradiography is a time-consuming procedure which relies upon reduction of silver ions to form silver grains in a photographic emulsion; and scintillation counting, the other detection technique, requires expensive equipment and a certain amount of delay as well. Furthermore, radioisotopes require special handling for safety reasons. Some radioactive isotopes, such as .sup.125 I, have relatively short shelf-lives, which further limit their usefulness in a clinical diagnostic setting.
In non-radioactive labelling systems, a probe is "labelled" with a reporter group which is associated with a signal to enable detection. A reporter is an agent which is used to associate a signal with a probe for indicating the presence or location of the probe. The signal itself, which is directly perceptible, may be generated by a separate or separable signal molecule. A label is properly a type of reporter which incorporates a signal.
One approach to the attachment of labels to probes is described in Ward, et al., European patent application No. 63,879. Ward discloses the preparation of probes having a biotin reporter molecule covalently attached to a purine or pyrimidine ring. Selected biotinylated purines and pyrimidines are then directly incorporated within the phosphodiester backbone of the nucleic acid of the probe by enzymatic means. However, enzymatic techniques are costly and difficult to perform.
Other approaches link a label to a probe by way of a protein. Single-stranded polio virus RNA is naturally linked to a protein which may be reacted with the N-hydroxysuccinimidyl ester of biotin to obtain an RNA probe having a biotinylated reporter group detectable by specific attachment of avidincoated spheres. Richards, et al., Proc.Natl.Acad.Sci. (USA), 76: 676-680 (1979). Similarly, biotin-labelled cytochrome c may be coupled to RNA by reaction in the presence of formaldehyde and thereafter labelled with avidin-coated spheres. Manning, et al., Chromosoma (Berl.), 53: 107-117 (1975). Nevertheless, because not all nucleic acids desired to be labelled are naturally associated with proteins and because the location and amount of cytochrome c binding to a nucleic acid is not readily predictable, it is desirable to have a chemical synthetic technique for end-labelling.
In one such chemical synthetic technique, nucleic acids are converted to 3'-aldehydes by oxidation and condensed with alkyldiamines or polyamines to provide a reporter group for the attachment of biotin. Broker, et al., Nucleic Acids Res., 5: 363-384 (1978). Similarly, aldehydes generated by the periodate oxidation of nucleic acids may be used to couple fluorescent labels to the nucleic acids. Bauman, et al., J. Histochem.Cytochem., 29: 227-237 (1981). However, it is desirable to have a technique for attaching reporter groups to polynucleotides attached to a support in an automated process for nucleic acid synthesis.
In yet another approach to 5' labelling, biotin is converted to 2-(biotinylamido)ethanol and condensed to a phosphorylated, polymer-supported nucleotide. The condensation of the aminoethanol derivative of biotin to the 5' hydroxyl group of a ribose ring gives a stable phosphodiester bond upon deprotection of the nucleotide. Kempe, et al., Nucleic Acids Res., 13: 45-57 (1985). Nevertheless, specific reporter groups are attached by this approach so that the approach does not permit preparation of an oligonucleotide with a reactive functionality which may later be used to attach a variety of desired reporter groups.
Nucleotides in solution have been amine-functionalized by condensation with protected 6-amino-1-hexanol phosphate. Barker, et al., J.Biol.Chem., 22: 7135-7147 (1972). However, these procedures are difficult to perform and have not been integrated with solid-phase synthesis.
In another approach to binding nucleotides to supports, in the purification of nucleases by affinity chromatography, single nucleotides, 3'-derivatized with p-aminophenol are attached to a gel matrix by a linker. The linker is formed by attaching 3,3' diaminodipropylamine to the matrix using cyanogen bromide and azide. The resulting amine-functionalized gel is treated with succinic anhydride and then coupled to the amine-functionalized nucleotide. Cuatrecasas, J.Biol.Chem., 12: 3059-3065 (1978). Nevertheless, the manufacture of the amine-functionalized nucleotide itself has been performed in solution by tedious procedures. See, e.g., Barker, et al., J.Biol.Chem., 22: 7135-7147 (1972).
In an approach to 5' labelling, a 5-aminoalkyl phosphoramidate derivative of a kinased, unprotected oligonucleotide is prepared in solution. Kination of a deprotected oligonucleotide is accomplished by T4 polynucleotide kinase. The kinased oligonucleotide is reacted with an imidazole and a diamino alkane. The resulting 5'-aminoalkyl phosphoramidite DNA is reacted with biotin-N-hydroxysuccinimidyl ester in potassium N,N-dimethylformamide to produce a biotin-labelled oligodeoxyribonucleotide. Chollet, et al., Nucleic Acids Res., 13: 1529-1541 (1985). Although this method makes use of an oligonucleotide synthesized using the solid-phase phosphoramidite method or the solid-phase phosphotriester method, the oligonucleotide is deprotected, detached from the solid phase, isolated, and purified before functionalization. After functionalization, a second isolation and purification is required. Because each isolation and purification entails losses and is time-consuming, it is desirable to functionalize a polynucleotide during solid-phase synthesis. Furthermore, the kination reaction suffers from the problems of cost and difficulty generally associated with enzymatic procedures.
Therefore, there is a need for a method and composition for the generic attachment of reporter groups to polynucleotides undergoing solid phase synthesis.